Prog Biomater (2016) 5:223–235
DOI 10.1007/s40204-016-0060-8
ORIGINAL RESEARCH
Fabrication and characterization of biosilver nanoparticles loaded
calcium pectinate nano-micro dual-porous antibacterial wound
dressings
Robin Augustine1,2 • Anitha Augustine3 • Nandakumar Kalarikkal1,4
Sabu Thomas1,5
•
Received: 16 August 2016 / Accepted: 7 November 2016 / Published online: 2 December 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Development of materials for medical applications using biologically derived materials by green
approaches is emerging as an important focus in the present
healthcare scenario. Herein the first time, we report the
plant extract mediated ultra-rapid biosynthesis of silver
nanoparticles using whole plant extracts of Biophytum
sensitivum. Synthesized nanoparticles were immobilized in
nano-micro dual-porous calcium pectinate scaffolds for
wound dressing application. Pectinate wound dressings
containing silver nanoparticles have shown excellent
antibacterial property and exudate uptake capacity while
being biocompatible to the human cells.
Keywords Silver nanoparticles Biophytum
Biosynthesis Pectinate Wound dressings
& Robin Augustine
robin@robinlab.in; robinaugustine9@gmail.com
& Sabu Thomas
sabuchathukulam@yahoo.co.uk
1
International and Inter University Centre for Nanoscience and
Nanotechnology, Mahatma Gandhi University, Kottayam,
Kerala 686560, India
2
School of Nano Science and Technology, National Institute
of Technology Calicut, Calicut, Kerala 673601, India
3
Department of Chemistry, Bishop Kurialacherry College for
Women, Amalagiri, Kottayam, Kerala 686561, India
4
School of Pure and Applied Physics, Mahatma Gandhi
University, Kottayam, Kerala 686560, India
5
School of Chemical Sciences, Mahatma Gandhi University,
Kottayam, Kerala 686560, India
Introduction
Skin is the largest organ of the body which performs many
crucial roles for instance as a barrier against exogenous
substances including pathogens and mechanical stresses
(Augustine et al. 2014a). Skin is always in direct contact
with the external environment which make them highly
susceptible to damage and/or injury (Fuchs 2016). Thus,
fast repair of the skin after an injury is necessary. Now-adays, polymeric wound dressings were developed to act as
analog of the skin by performing many of the functions of
natural skin like exudate management capacity, preventing
microbial invasion and thermal protection (Miraftab et al.
2003; Augustine et al. 2014b, 2015a, b; Xu et al. 2015).
Hydrogels like alginate and pectinate can manage the
excessive exudate produced in the wound site and can act
as a thermal barrier (Lloyd et al. 1998). However, additional strategies should be adopted to prevent the bacterial
invasion and colonization in the wound. Incorporation of
antimicrobial agents in the wound dressing is a robust
approach to overcome wound infections (Augustine et al.
2014c). Antibiotics have been tried as antibacterial agents
in polymeric wound dressings to avoid bacterial colonization in the wound (Unnithan et al. 2012). Due to the bacterial drug resistance and less chemical stability of the
antibiotics, relatively stable novel materials should be
exploited as antibacterial agents in wound dressings.
Silver nanoparticles (AgNP) are well established for
their inhibitory activity against wide range of pathogenic
microorganisms (Rai et al. 2009; Augustine and
Rajarathinam 2012). There are many methods for the
synthesis of metallic nanoparticles (Sundaram et al. 2012).
Green approaches like biological synthesis routs have been
adopted to enhance the biocompatibility of produced
nanoparticles (Mohanpuria et al. 2008). In such green
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Prog Biomater (2016) 5:223–235
methods, instead of chemical reducing agents biologically
derived substances are used to convert silver ions to AgNP.
Natural plant extracts have been emerged as biological
reducing agents in green routes for the synthesis of AgNP
(Saravanan et al. 2011; Chandran et al. 2006; MubarakAli
et al. 2011; Mollick et al. 2015; Latha et al. 2016; Jadhav
et al. 2016). The major advantage of using extracts of the
plants (whole plant, leaves, roots etc.) for AgNP synthesis
is that they are locally available, safe, and the increased
biocompatibility of the resulting AgNPs (Park 2014). It has
been reported that the phytochemicals are involved directly
in the reduction of the silver ions and the formation of
AgNPs (Jha et al. 2009). The main phytochemicals
involved in the reduction of silver salts are flavones, terpenoids, ketones, amides, aldehydes and carboxylic acids
(Prabhu and Poulose 2012). Biophytum sensitivum is a
plant in the Oxalidaceae family widely distributed in India,
Nepal and in other south-east Asian countries and is used
for medicinal purposes (Sakthivel and Guruvayoorappan
2012). Phytochemical analysis has shown that this plant
contains various medicinal biochemicals which include
amentoflavone, cupressuflavone and isoorientin. Biophytum plant extract and its bioactive compounds have been
known to possess antioxidant, anti-inflammatory, antibacterial, antitumor, radioprotective, antimetastatic, chemoprotective,
antiangiogenic,
wound
healing,
immunomodulatory, anti-diabetic, and cardioprotective
activity (Lee et al. 2009; Sakthivel and Guruvayoorappan
2012; Wilsky et al. 2012; Lee et al. 2013). Presence of
biomolecules from this medicinal plant may enhance the
biological properties of synthesized nanomaterials. Our
group has demonstrated that biologically synthesized
AgNPs using extracts of black pepper shows superior
antibacterial property (Augustine et al. 2014d, 2015c).
High quality colloidal suspensions of AgNP should show
relatively narrow size distributions, high uniformity in
shape and excellent dispersibility to eliminate aggregation.
Pectin is a natural, linear, heterogeneous polysaccharide
industrially extracted from citrus fruit peels and apple
pomace (May 1990; Augustine et al. 2015d). Pectin mainly
consists of D-galacturonic acid (GalA) units joined in
chains by means of a (1–4) glycosidic linkages with
alternating side chains of a (1–4) D-gaIactose and D-arabinose (Augustine et al. 2013). The unique gel forming
ability of polyuronates in the presence of calcium ions
makes them ideal for drug delivery and wound dressing
applications (Augustine and Rajarathinam 2012). The
divalent calcium ions and the negatively charged carboxylate groups of the polyuronates forms intermolecular
crosslinks resulting in an ‘‘egg-box’’ structure of rigid gel
networks which are relatively stable under physiological
conditions. Low methoxy (LM) pectins gels in the presence
of divalent cations, such as Ca2? (Augustine et al. 2015d).
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There are many advantages for pectinate based wound
dressings like excellent exudate uptake capacity and
biocompatibility. However, such dressings are highly
prone to bacterial colonization and they could not prevent
invasion of such pathogenic bacteria to the wound (Mishra et al. 2011; Tummalapalli et al. 2016). Incorporation
of biosynthesized AgNP in the calcium pectinate (CaP)
wound dressing would be a novel approach to overcome
this challenge. Our previous study demonstrated that
incorporation of AgNP within the range of 0.25 and
1 wt% in the polymer matrix could provide satisfactory
antibacterial property to the wound dressings (Augustine
et al. 2015c).
Our aim in this study is to develop a porous flexible
calcium pectinate/silver nanocomposite (CaP-AgNP)
hydrogel wound dressing with excellent exudate management capacity, biocompatibility and antimicrobial properties. The advantages of using this dressing are; absorption
of wound exudates, prevention of wound infection, retention of optimum moisture environment, permeation of
gasses and fast wound healing. The wound healing ability
and antibacterial activity of AgNP can be further enhanced
due to the presence of phytochemicals from Biophytum.
Thus, CaP-AgNP nanocomposite membranes will function
as ideal wound dressings with excellent antibacterial
property and exudate uptake capacity while being biocompatible to the human body.
Materials and methods
Materials used
Pectin (extracted from apple, Mw 30,000–100,000,
70–75% esterification) and silver nitrate of analytical grade
quality were purchased from Sigma-Aldrich was used as
the starting material without further purification. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), trypsin/EDTA (ethylenediaminetetraacetic
acid) solution, Mueller–Hinton agar and Nutrient broth
media were purchased from HiMedia, India. Biophytum
whole plants were freshly collected from the agricultural
field near Poothampara, Kozhikode, India.
Synthesis of silver nanoparticles
Hundred gram of freshly collected Biophytum plants were
cleaned in tap water, subsequently in deionized water and
grinded using mortar and pestle. 100 ml of deionized water
was poured into the slurry and filtered through Whatman
No. 1 filter paper. Filtrate was centrifuged at 5000 rpm to
completely remove the solid substances. Fresh supernatant
was used for the synthesis of silver nanoparticles.
Prog Biomater (2016) 5:223–235
Various concentrations of aqueous solutions (1, 2, 3, 4
and 5 mM) of silver nitrate (AgNO3) were prepared in
double distilled water and used for the synthesis of silver
nanoparticles. 100 ml silver nitrate solution was taken in a
round bottom flask and heated to boiling on a magnetic
stirrer. Light-mediated reduction of silver nitrate was
avoided by covering the flask with aluminum foil. 10 ml of
the Biophytum plant extract was added drop wise into the
silver nitrate solution. During this process, the solution was
stirred vigorously. Within 1 min, the color change was
evident (pale orange to pale red). Then, it was removed
from the hotplate and stirred for 1 h to be cooled to room
temperature. A small portion of the obtained nanoparticle
suspensions were used for UV–Visible spectroscopic
analysis. Remaining part was centrifuged at 12,000 rpm
several times in deionized water and finally in alcohol to
get pure silver nanoparticles.
UV–Vis spectroscopic analysis of AgNP
The optical properties of colloidal solution of AgNP were
evaluated using a Shimadzu double-beam spectrophotometer between 200 and 600 nm at a resolution of 1 nm.
FTIR (Fourier-transform IR) analysis of silver
nanoparticles
FTIR analysis of the dried AgNP samples was carried out
using Perkin Elmer, Spectrum 400 spectrophotometer to
ensure the formation of silver nanoparticles from silver
nitrate. FTIR measurement is useful to determine the
presence of bioactive molecules, which may be responsible
for stabilization of AgNPs by acting as capping agents.
X-ray diffraction (XRD) analysis of silver
nanoparticles
XRD was recorded in the 2h range of 20–90° using D8Advance of Bruker (Germany), of CuKa radiation with the
energy 8.04 keV and wavelength 1.54 A°. The current was
25 mA and applied voltage was 40 kV. Obtained data were
compared with the ICDD PDF2 powder diffraction database (International Centre for Diffraction Data 2007).
Transmission electron microscopic (TEM) analysis
of AgNP
JEOL JEM 2100 high resolution TEM was used to image
the AgNP to understand the morphology and size distribution of synthesized AgNPs. The samples for TEM
analysis were prepared by air-drying drops of dilute solu-
225
tions of colloidal suspensions of AgNP on carbon films.
ImageJ software was used to measure the individual particle size. Particle size was measured for 50 particles for
each sample, particle size distribution curves were drawn
and the average particle sizes were calculated.
Antimicrobial activity of AgNPs
The Kirby–Bauer disc diffusion method was used to
determine the growth inhibition of bacteria by the synthesized AgNPs. The bacterial strains Escherichia coli (ATCC
12228) and Staphylococcus aureus (ATCC6538-P) were
used as representatives of Gram-negative and Gram-positive bacteria, respectively. Both the bacteria were cultured
separately in Nutrient Broth medium at 37 °C in an incubator and prepared to the turbidity equivalent of 0.5
McFarland standards (McFarland 1907). Then, 100 ll of
the bacterial suspension was spread on the Mueller–Hinton
agar plates. Sterile filter paper discs with 6 mm diameter
(HiMedia, Mumbai) were impregnated with AgNP synthesized at various silver nitrate concentrations so that to
get a final AgNP concentration of 10 lg/disc. A standard
antibiotic disc was used as positive control (Ciprofloxacin,
30 lg/disc). Both the paper discs containing AgNP and the
controls were then placed on the surface of the Mueller–
Hinton agar culture plates which were swabbed with the
bacteria. The culture plates were incubated for overnight in
an incubator at 37 °C. The diameters of the inhibition
zones were measured in millimeters (mm). The experiment
was repeated for three times to get an average value and
expressed as mean ± S.D.
Fabrication of CaP-AgNP wound dressing
For the fabrication of biosynthesized AgNP containing
porous CaP scaffolds, lyophilization technique was used.
Based on the preliminary results of antibacterial activity of
AgNPs, nanoparticles synthesized using 2 mM silver
nitrate solution was used for incorporating in CaP scaffolds. Pectin solution (1 w/v%) was prepared by dissolving
a known quantity of pectin in 50 ml deionized water with
continuous stirring in a magnetic stirrer to form a transparent solution. Biosynthesized AgNP were properly dispersed in the above solution to make CaP-AgNP dressings.
CaP-AgNP containing 0.25 wt% (CaP-AgNP-0.25),
0.5 wt% (CaP-AgNP-0.5) and 1 wt% (CaP-AgNP-1) of
AgNP were prepared. A bare sample without silver
nanoparticle incorporation was also maintained (CaP).
These solutions were poured into petri dishes (5 mm
thickness) and dried in hot air oven (60 °C) for overnight to
form films. The films were crosslinked with 4% calcium
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Prog Biomater (2016) 5:223–235
chloride solutions (for 1 h) and washed with distilled water
for several times to remove the residual CaCl2. The films
were lyophilized individually for 48 h.
The fabricated CaP-AgNP nanocomposite scaffolds
were characterized using techniques like SEM and XRD
analyses (as described in the previous section). The exudate
uptake capacity, antimicrobial properties and the biocompatibility of the fabricated scaffolds were also evaluated.
Scanning electron microscopy (SEM) of CaP-AgNP
dressings
A Zeiss Ultra Plus High Resolution FEG-SEM (Zeiss,
Germany), operated at 4.0 kV, using an in-lens secondary
electrons (SE) detector was used for the morphological
analysis of the CaP and CaP-AgNP membranes. Prior to
the analysis, the samples were coated with gold/platinum
alloy.
XRD analysis of CaP-AgNP dressings
XRD patterns of CaP, CaP-AgNP wound dressings were
recorded in the 2h range of 5°–90° using D8-Advance of
Bruker (Germany) of CuKa radiation with the energy
8.04 keV and wavelength 1.54 A°. The applied voltage
was 40 kV and current was 25 mA.
Swelling study of CaP-AgNP dressings
Ability of the CaP-AgNP dressings to absorb water from
phosphate buffered saline (PBS, pH-7.4) and swell themselves was studied to understand the diffusion of wound
exudates into the dressing that is essential for proper exudate management in the wound bed. Previously weighed
dressings were immersed in PBS solution in pre-weighed
containers for known intervals of time. The PBS solution
was completely discarded at specific intervals and wet
weight was measured. Percentage of swelling was calculated using the formula:
Percentage swelling ¼ ðfinal weight
initial weightÞ
=initial weight 100:
CaP-AgNP-1 were used. The discs of lyophilized membranes were cut into 6 mm diameter and placed on the
surface of the inoculated MHA plates. The plates were
incubated at 35 °C overnight to get a confluent lawn of
bacterial growth. Gentamicin antibiotic discs containing
10 lg/disc were used as positive controls. The sensitivity
of the microorganisms to the membranes was determined
by measuring the diameter of inhibitory zones on the agar
surface around the discs. All the tests were carried out in
triplicate. The diameters of the inhibitory zones were
measured in millimeters.
Determination of in vitro biocompatibility
of the CaP-AgNP dressings
Cell viability on CaP and CaP-AgNP wound dressings
were determined by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. L929 fibroblast
cell line was obtained from NCCS Pune and was maintained in Dulbecco’s modified eagles media (HIMEDIA)
supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and grown to confluency at 37 °C in 5% CO2 (NBS,
EPPENDORF, GERMANY) in a humidified atmosphere in
a CO2 incubator. The cells were trypsinized (500 ll of
0.025% Trypsin in PBS/0.5 mM EDTA solution (Himedia)) for 2 min and passaged to tissue culture flasks in
complete aseptic conditions. Scaffolds with 1 cm2 size
were sterilized and immersed in cell free media for 24 h.
Trypsinzed cells (50,000/cm2) were added on the surface of
samples and were allowed to grow for 24 h followed by
MTT assay. Briefly, the cultured cells or tissues were
washed thoroughly with PBS and then incubated in MTT
solution (0.5 mg/ml MTT in PBS) for 3 h at 37 °C with 5%
CO2 supply. Subsequently, the solution was aspirated and
the insoluble formazan product was solubilized with acidified iso-propanol. Incubated at room temperature for
30 min until the cell got lysed and a purple color was
obtained. The optical density was then determined at
540 nm using a multi-well plate reader (LISASCAN,
Erba). Percentage of cell viability was calculated using the
following equation:
% Viability ¼ ðOD of test=OD of controlÞ 100:
Antimicrobial activity of CaP-AgNP dressings
In vitro antibacterial activity of CaP and CaP-AgNP
dressings was evaluated by disc diffusion method according to the National Committee for Clinical Laboratory
Standards (NCCLS 2001). The procedure adopted for this
experiment was similar to the antibacterial testing used for
AgNP except that instead of paper discs containing silver
nanoparticle CaP, CaP-AgNP-0.25, CaP-AgNP-0.5 and
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Statistical analysis
All the experiments were carried out in triplicate and the
results were expressed as mean ± standard deviation.
Statistical significance between different groups was
determined by Student’s t test. A P value less than 0.05 was
considered as statistically significant.
Prog Biomater (2016) 5:223–235
Results
Visible color change and UV absorbance
during the formation of AgNP
Visual color change will give a preliminary information
regarding the formation of AgNPs. As the AgNPs are
formed, the color of the solution changed from colorless to
pale orange to brick red which indicates the formation of
AgNPs (Fig. 1a). It is well accepted that difference in
localized surface plasmon resonance (LSPR) of AgNPs
with particle size makes a variation in the optical properties
(Sherry et al. 2005). A very pale orange color was observed
for the AgNPs synthesized at a silver nitrate concentration
of 1 mM. There was a considerable increase in the redness
of the solution when the concentration of silver nitrate was
increased up to 5 mM. Corroborating the results of previous studies, as the concentration of silver nitrate increases,
aggregation of formed silver ions occurs and which leads to
the formation of larger sized AgNPs (Augustine et al.
2014c).
227
The UV–Visible absorption spectra of biosynthesized
AgNPs are shown in Fig. 1b. Characteristic absorption
maxima of silver nanoparticles can be observed in between
300 and 600 nm. For 1 mM solution, AgNPs have shown
absorbance maximum at 398 nm, 2 mM solution has
absorbance maximum at 402 nm, 3 mM has at 404 nm,
4 mM has at 406 nm, and 5 mM solution has at 407 nm.
These range (398–406 nm) of absorption maxima indicate
the presence of AgNPs with a particle size below 20 nm
(Pillai and Kamat 2004). Localized surface plasmon resonance (LSPR), which is a result of collective oscillations of
a nanoparticle’s conduction band electrons, is the reason
for the variation in the optical properties of nanoparticles
(Sherry et al. 2005). Characteristics of the surface plasmon
absorption depend on the size and shape of the nanoparticles (Wang et al. 2007). The absorption maxima shift
towards red with increasing molar concentration of silver
nitrate which is an indication of the increase in particle size
of AgNPs (Rai et al. 2006; Song and Kim 2009; Fayaz
et al. 2009).
FTIR analysis of AgNPs
The FTIR spectra of unreduced silver nitrate and AgNPs
after the reduction and stabilization by Biophytum extract
were taken and presented in Fig. 2. FTIR analysis will help
Fig. 1 Photographic image showing the color variation of AgNPs
synthesized using various concentrations of silver nitrate solutions
(a). UV visible spectra of AgNPs synthesized using various concentrations of silver nitrate (b)
Fig. 2 Representative FTIR spectra of biosynthesized AgNPs and
silver nitrate
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Prog Biomater (2016) 5:223–235
X-ray diffraction (XRD) analysis of AgNPs
The structure of prepared AgNP has been investigated by
X-ray diffraction (XRD) analysis. XRD patterns of the
synthesized AgNPs are shown in the Fig. 3. The obtained
XRD patterns have indicated the successful formation of
AgNPs. The data shows diffraction peaks at 2h = 38.2°,
44.05°, 64.7° and 77.8° which can be indexed to (111),
(200), (220) and (311) crystalline planes of cubic silver
(PDF No. 04-0783). Obtained results clearly indicate that
the AgNP formed by the reduction of Ag? ions by the
biophytum extract are crystalline in nature. The broad
nature of the XRD peaks could be attributed to the very
nano size of the particles. Average particle size has been
calculated using Debye–Scherrer formula:
123
(a)
(b)
Intensity (a.u)
to identify the biomolecules present in Biophytum extract
which was present over AgNPs. While comparing the FTIR
spectra of unreduced silver nitrate and biosynthesized
AgNPs, disappearance of certain peaks and appearance of
some new peaks was observed. Corroborating to the previous studies, absorbance bands in pure silver nitrate were
observed in the region of 450–750 cm-1 and were 1747,
1286, 790, 730 and 635 cm-1 which are due to the presence of nitro compounds (Augustine and Rajarathinam
2012, 2014c). A broad peak at 1286 cm-1 which was
present in the spectrum of silver nitrate was not observed in
the spectrum of AgNPs. It indicates the loss of nitro group
from silver species during the formation of AgNPs.
The FTIR spectrum of AgNPs showed a broad peak in
between 2800 and 3500 cm-1 and distinct peaks at 1731,
1594, 1371, 1218, 1030 and 808 cm-1. The broad
absorption peak in between 2800 and 3500 cm-1 represents the presence of OH functional groups. Peak at
1731 cm-1 is an indication of C=O stretch and probably
due to aromatic esters present in B. sensitivum (Amentoflavone, Cupressuflavone and/or Isoorientin). Ketones
show their carbonyl C=O stretch at 1740–1730 cm-1, but
also exhibit their characteristic absorption at
1300–1000 cm-1 from the couplings of C–O and C–C
stretches. Thus, the presence of C–O stretch in between at
1218 and 1030 cm-1 may be due to the covalent linking of
C=O groups containing flavonoids to the nanoparticles.
The peak 1594 cm-1 is due to carbon–carbon stretching
vibrations in the aromatic rings of the flavonoids attached
to the nanoparticles.
These observations indicate the presence and binding of
certain biomolecules with AgNPs. It may be due to the
binding of one or more flavanones (Amentoflavone,
Cupressuflavone, Isoorientin etc.) and/or amide-containing
alkaloids which is present in Biophytum to the synthesized
AgNPs. This may contribute to the stability of synthesized
AgNPs (Augustine et al. 2014c).
(c)
(d)
311
*
(e)
20
111
*
200
**
30
220 *
40
50
60
2 Theta (Degrees)
70
80
Fig. 3 XRD patterns of AgNPs synthesized using 1 mM (a), 2 mM
(b), 3 mM (c), 4 mM (d) and 5 mM (e) silver nitrate solutions
D ¼ 0:9k=b Cosh;
where ‘k’ is wave length of X-ray (0.1541 nm), ‘b is
FWHM in radians, ‘h’ is the diffraction angle and ‘D’ is the
diameter (size) of the synthesized nanoparticles. The particle size was found to be 7.62, 8.52, 8.61, 9.62 and
12.47 nm for AgNPs which are synthesized using 1, 2, 3, 4
and 5 mM silver nitrate solutions, respectively. The unassigned peaks denoted by (*) could be due to the presence of
crystalline organic phases from the plant extract. Awwad
et al., observed such additional peaks in the XRD spectrum
of green synthesized AgNPs using carob Olea europaea
leaf extract (Awwad et al. 2012) and carob leaf extract
(Awwad et al. 2013). Sathyavathi et al. (2010) and Khalil
et al. (2014) also observed such additional peaks in the
XRD pattern of biosynthesized AgNPs using Coriandrum
sativum leaf extract and olive leaf extract, respectively.
TEM imaging of AgNPs
TEM is a powerful tool to understand the morphology as
well as particle size of nanomaterials. TEM images of
AgNPs obtained at various silver nitrate concentrations are
given in Fig. 4.
In general, the synthesized AgNPs were spherical in
morphology without forming any agglomerates. The
average particle size at 1 mM silver nitrate solution concentration was 7.4 nm. At a silver nitrate concentration of
2 mM, almost similar morphology was obtained as in the
case of 1 mM solution but the average particle diameter
increased to 8.2 nm (Fig. 4b). When the silver nitrate
concentration was further increased, the size of the
nanoparticles was also tends to increase. At 3 mM silver
nitrate concentration, AgNPs with an average particle size
of 8.8 nm were obtained (Fig. 4c). From Fig. 4d, it is clear
that at 4 mM silver nitrate concentration, the average
Prog Biomater (2016) 5:223–235
229
Fig. 4 TEM images of AgNPs synthesized using silver nitrate solutions of 1 mM (a), 2 mM (b), 3 mM (c), 4 mM (d) and 5 mM (e). Graphs
shows the particle size distribution of each sample based on TEM images (b)
particle size was 9.1 nm and the morphology was still
comparable with those synthesized using 1, 2 and 3 mM
silver nitrate solutions. The particle size distribution was
broadened for the AgNPs synthesized using 5 mM silver
nitrate solution and the average particle size was 11.4 nm.
Previous studies showed that small nanoparticles formed in
the solution themselves can act as nucleation centers, and
thus at higher concentration of metal ions these seeds will
grow further and hence large sized nanoparticles will be
obtained (Mallik et al. 2001). A similar mechanism could
be proposed here also.
Table 1 Diameter of inhibitory zone
Evaluation of antibacterial property of AgNP
synthesized using 1, 2 and 3 mM silver nitrate solutions
showed superior antibacterial property than those synthesized using 4 and 5 mM silver nitrate solutions (P \ 0.05)
against both bacteria. Nanoparticles prepared using low
concentrations of silver nitrate were more effective to
inhibit both E. coli and S. aureus. This can be due to the
fact that antibacterial activity of AgNPs was found to be
dependent on the size of the nanoparticles and as the size
increases the antibacterial activity decreases (Panáček et al.
2006). From the morphological features of the AgNPs, it
was evident that AgNPs synthesized using 4 mM and
5 mM silver nitrate solutions were comparatively larger in
size. All the synthesized nanoparticles have shown more
The antibacterial activity of the AgNPs was evaluated by
observing their inhibitory activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria by
disc diffusion method (Kirby–Bauer method). The results
of the antimicrobial testing are shown in Table 1. From the
table, it is evident that all the AgNPs has shown excellent
antibacterial activity against both S. aureus and E. coli.
While comparing the inhibitory zone diameters of AgNPs
synthesized using 1, 2 and 3 mM silver nitrate solutions
against S. aureous and E. coli, there was no considerable
statistical difference (P [ 0.1). However, the AgNPs
Silver nitrate concentration (mM)
Inhibitory zone (mm)
E. coli
S. aureus
1
13.4 ± 1.2
11.1 ± 1.7
2
3
13. 9 ± 0.9
12.5 ± 1.1
11.5 ± 1.1
11.2 ± 0.7
4
10.8 ± 0.6
10.5 ± 1.4
5
10.5 ± 1.4
9.6 ± 0.9
30 ± 2.6
29.5 ± 1.8
Ciprofloxacin
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Prog Biomater (2016) 5:223–235
Fig. 5 SEM images of CaP (a,
e) and CaP-AgNP-0.25 (b, f),
CaP-AgNP-0.5 (c, g) and CaPAgNP-1 (d, h)
antibacterial activity against E. coli than S. aureus as
reported by other workers (Shrivastava et al. 2007). The
reason for such an observation could be explained in terms
of the difference in the cell wall structure of these bacteria.
Moreover, the biomolecules present on AgNPs enhances
the antibacterial efficacy due to the antibacterial property
of these molecules. Antibacterial activity of B. sensitivum
on both Gram-negative and Gram-positive bacteria was
already reported (Natarajan et al. 2010; Sakthivel and
Guruvayoorappan 2012). Based on the results obtained
from the disc diffusion technique, it was clear that the
synthesized AgNPs can successfully inhibit bacterial proliferation and hence it can be used for the development of
materials where antibacterial property is essential.
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Morphological features of CaP-AgNP membranes
Morphological features of the fabricated dressings were
evaluated by SEM analysis and given in Fig. 5. From the
figure, it is clear that the fabricated scaffolds were nanomicroporous in structure. This kind of dual-porous structures are advantageous for wound dressings in the sense
that a large degree of porosity is required for the gas
exchange and immediate swelling (Mi et al. 2001). Addition of 0.25 wt% of AgNP did not change the morphology
of the dressings and were similar to the bare CaP dressings
in terms of nano and microporosity. However, CaP-AgNP0.5 and CaP-AgNP-1 showed much variation than bare
CaP dressings in morphological features. They possessed
Prog Biomater (2016) 5:223–235
231
8000
80
6000
Swelling (%)
Intensity (a.u)
60
(a)
40
(b)
20
4000
CaP
CaP-AgNP-0.25
CaP-AgNP-0.5
CaP-AgNP-1
2000
(c)
0
(d)
0
20
40
60
2 Theta (degrees)
0
80
Fig. 6 XRD spectra of bare CaP (a), Cap-AgNP-0.25 (b), CaPAgNP-1 (c) and AgNP (d)
1
2
3
Time (h)
4
5
Fig. 7 Exudate uptake capacity of CaP and CaP-AgNP-0.25, CaPAgNP-0.5 and CaP-AgNP-1 at various time intervals in terms of
percentage of swelling
XRD analysis of the scaffolds confirmed the presence of
AgNP in the CaP-AgNP dressings. Bare CaP dressings
were relatively amorphous in nature as evident from the
week peaks observed in the XRD pattern (Fig. 6). Three
broad peaks were found at 2 theta 13.7, 30 and 55 degrees.
These peaks are due to the egg-box junction zones of
calcium pectinate (Guo et al. 2014). Li et al. (2007) also
reported comparable XRD patterns of the egg-box junction
zones in the case of calcium alginate. Apart from these
peaks, the nanocomposites showed the characteristic XRD
patterns of AgNP also. In the case of CaP-AgNP-0.25,
though these patterns were present, the intensity was very
low to be distinguished from the background. However,
when the concentration of AgNP increased in the
nanocomposites, well distinguishable sharp diffraction
patterns of AgNP were observed.
and *5780%, respectively, with statistically significant
difference (P \ 0.05). At second hour of immersion, CaP
and CaP-AgNP-0.25 have reached a swelling of *7360
and *6415%, respectively. This trend was continued even
at fifth hour immersion. CaP-AgNP-0.5 and CaP-AgNP-1
showed a maximum swelling after 1 h immersion in PBS
which were *4360 and *3280%, respectively. From 1 h
onwards, all the scaffolds were shown statistically significant variation in swelling each other (P \ 0.05). Thus, the
swelling of all the scaffolds increased with increasing
immersion time up to 1 h. However, a further increase in
the immersion time did not produce any significant variation in swelling. There was no effect of AgNP on the
swelling of CaP up to 1 h of immersion in PBS. In contrast,
presence of AgNP in the CaP has a significant effect on the
swelling of CaP after 1 h of immersion (P \ 0.05). As the
percentage of AgNP in the CaP was increased, the percentage of swelling was decreased. Polymers in general
and especially hydrogels shows a reduction in swelling in
water when nanoparticles are incorporated in them (Fan
et al. 2013).
Exudate uptake capacity of CaP-AgNP
Antibacterial property of CaP-AgNP scaffolds
The exudate uptake capacity of CaP, CaP-AgNP-0.25,
CaP-AgNP-0.5 and CaP-AgNP-1 scaffolds after immersion
in PBS (pH-7.4) at 37 °C up to 5 h were studied and the
results are shown in Fig. 7. During the first 30 min of
immersion in PBS, the swelling of the CaP and CaP-AgNP0.25 scaffolds did not show any significant difference but
both showed *4630% of swelling (P [ 0.05). Similarly,
CaP-AgNP-0.5 and CaP-AgNP-1 scaffolds did not show
any significant variation in swelling up to 30 min. However, in the case of CaP and CaP-AgNP-0.25, from 1 h of
immersion period onwards, the value increased to *6815
The antibacterial activity of the CaP-AgNPs containing various concentrations of AgNP was evaluated by disc diffusion
technique against a common Gram-negative (E.coli) and
Gram-positive (S.aureus) bacteria and the results are shown in
Fig. 8 and Table 2. From these results, it is evident that fabricated scaffolds containing AgNP have shown good inhibitory activity against both E. coli and S. aureus. The CaP
membranes did not show any antibacterial activity against the
tested bacteria. CaP-AgNP-0.25 has showed statistically significant antibacterial activity with an inhibitory zone diameter
of 8.7 ± 0.6 against E.coli but it does not show any activity
more micropores
architectures.
and
some
special
nanosurface
XRD analysis of CaP-AgNP
123
232
Prog Biomater (2016) 5:223–235
Fig. 8 Plates showing the
antibacterial activity of the
fabricated CaP (a), CaP-AgNP0.25 (b), CaP-AgNP-0.5 (c),
CaP-AgNP-1 (d) against E. coli
(plate A) and S. aureus (plate
B). Gentamicin discs were used
as the positive control (e)
Table 2 Inhibition zone diameter from disc diffusion method using
CaP and CaP-AgNP wound dressings on E. coli and S. aureus
Sample
Table 3 Percentage of cell viability by MTT assay
Sample
Percentage of viability (%)
Control
100 ± 0
Inhibition zone diameter (mm)
CaP
CaP-AgNP-0.25
E. coli
S. aureus
6.00 ± 0
6.00 ± 0
8.7 ± 0.6
6.00 ± 0
CaP-AgNP-0.5
CaP-AgNP-1
9.3 ± 0.2
11.2 ± 0.4
6.7 ± 0.5
7.8 ± 0.9
Gentamycin
24.2 ± 0.8
26.7 ± 0.5
against S. aureus. Similarly, CaP-AgNP-0.5 has shown an
inhibitory zone diameter of 9.3 ± 0.2 and 6.7 ± 0.5 against
E. coli and S. aureus, respectively. CaP-AgNP-1 showed an
inhibitory zone diameter of 11.2 ± 0.4 and 7.8 ± 0.9 against
E.coli and S. aureus, respectively. The antibacterial activity of
CaP-AgNP membranes was higher against E.coli than against
S. aureus (P \ 0.05). Previous studies also showed that silver
nanoparticles could more effectively inhibit E. coli than S.
aureus (Kim et al. 2011). This higher activity of CaP-AgNP
against E. coli might be due to the difference in cell walls
between Gram-positive and Gram-negative bacteria. Apart
from inherent bactericidal property of silver nanoparticles, the
presence of biomolecules over biosynthesized nanoparticles
enhances antibacterial efficacy of CaP-AgNP nanocomposite
membranes. Based on the results obtained from the disc diffusion technique, it is clear that the fabricated CaP-AgNP
scaffolds, especially CaP-AgNP-0.5 and CaP-AgNP-1 can
effectively inhibit bacterial colonization in wounds. Due to the
excellent antibacterial activity, they can be used for wound
dressing applications.
In vitro biocompatibility of the CaP-AgNP wound
dressings
The bare CaP and CaP-AgNP dressings containing various
concentrations (0.25, 0.5 and 1 wt%) of AgNP were
123
CaP
98 ± 3
CaP-AgNP-0.25
97 ± 2
CaP-AgNP-0.5
94 ± 4
CaP-AgNP-1
86 ± 7
evaluated for their cytotoxicity on L929 fibroblast cell lines
by MTT cell viability assay. The obtained results of this
study is given in Table 3. The viability of the L929 cells
cultured with the bare CaP (98 ± 3) with that of the cells
cultured with CaP-AgNP-0.25 (97 ± 2) and CaP-AgNP0.5 (94 ± 4) were very close. However, CaP-AgNP-1 has
shown a slight reduction in viability compared to other
samples (P \ 0.05). This corroborates the cytotoxic effects
of AgNPs to impair mitochondrial function, as reported by
other researchers (Burd et al. 2007; Foldbjerg et al. 2011).
The relative mitochondrial activity of CaP-AgNP-1 was
found to be 86 ± 7%. However, compared to the cytotoxic
effects reported by other workers, materials fabricated in
this study were superior in the sense that they were all
below the approved toxicity level. Moreover, Fig. 9, gives
direct evidence of biocompatibility of the fabricated
materials on L929 fibroblast cells. Cells grown at the
vicinity of the samples that contains AgNP were comparable to that of the control plates in terms of both cell
morphology and cell density. Many studies indicated that
the AgNPs and silver ions deleteriously affect mitochondrial functionality, and this is probably correlated to the
generation of ROS (AshaRani et al. 2008). Superior biocompatibility of the CaP-AgNP scaffolds might be due to
the presence of biologically derived molecules as capping
agents over the nanoparticles. The viability of the cells
cultured with all the CaP-AgNP scaffolds came between
*86 and *100%, demonstrating that all the AgNP
Prog Biomater (2016) 5:223–235
233
Fig. 9 Growth of L929
fibroblast cells in the presence
of fabricated scaffolds CaP (a),
CaP-AgNP-0.25 (b), CaPAgNP-0.5 (c), CaP-AgNP-1
(d) and a control plate (e). Dark
areas denoted by (asterisk)
represent the regions where the
samples were placed
containing scaffolds were apparently nontoxic to L929
cells, indicating their biocompatibility and potential uses
for wound coverage applications.
Based on the overall performance of the scaffolds,
especially the antibacterial performance and cell viability,
CaP-AgNP-0.5 can be considered as the optimum candidate for future studies. This sample showed considerable
antibacterial property against both Gram-negative and
Gram-positive bacteria while being relatively biocompatible to human cells.
Conclusion
In this study, we have first time demonstrated that using
Biophytum sensitivum plant extracts, silver nanoparticles
can efficiently be produced without the use of hazardous
and toxic reducing agents, stabilizing agents and solvents.
Silver nitrate solution was used as the precursor and
aqueous extracts of the medicinal plant, Biophytum as
reducing as well as stabilizing agent. The average particle
size of the silver nanoparticles has been found to depend on
the molar concentration of the silver nitrate solution used
for the synthesis. The average particle size was below
10 nm unless the concentration of the silver nitrate solution
was above 4 mM. FTIR analysis has shown the presence of
phytochemicals that were attached on the synthesized
nanoparticles. Synthesized nanoparticles were incorporated
in calcium pectinate wound dressings. These wound
dressings were nano and microporous in morphology and
shown excellent exudate uptake capacity. They were
effective against both E. coli and S. aureus while being
highly biocompatible to the human cells. Based on the
overall performance, calcium pectinate scaffolds containing 0.5 wt% AgNP can be considered as the optimum
formulation for future studies. Incorporation of these biologically acceptable silver nanoparticles with antibacterial
property in calcium pectinate scaffolds, makes this
approach potentially exciting for the commercial production of greener wound dressings.
Acknowledgements RA thanks Science Engineering Research Board
(SERB), New Delhi, India for National Postdoctoral Fellowship
(NPDF) (Reference Number PDF/2016/000499).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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